Intranasal immunization with detoxified lipooligosaccharide from nontypeable haemophilus influenzae or moraxella

ABSTRACT

The invention relates to intranasal immunization with detoxified lipooligosaccharide from nontypeable  Haemophilus influenzae  or  Moraxella catarrhalis.

RELATED APPLICATIONS

[0001] This application is a continuation and claims the benefit ofpriority of International Application No. PCT/US01/32331 filed Oct. 16,2001, designating the United States of America and published in English,which claims the benefit of priority of U.S. Provisional Application No.60/288,695 filed May 3, 2001, and for U.S. purposes only, PCT/US01/32331is a continuation-in-part of U.S. patent application Ser. No. 09/789,017filed Feb. 20, 2001, issued as U.S. Pat. No. 6,607,725, which is adivisional of U.S. patent application Ser. No. 08/842,409 filed Apr. 23,1997, issued as U.S. Pat. No. 6,207,157, which claims the benefit ofpriority of U.S. pat. Appl. No. 60/016,020 filed Apr. 23, 1996, andPCT/US01/32331 is also a continuation-in-part of U.S. patent applicationSer. No. 09/610,034 filed Jul. 5, 2000, pending, which is a continuationof Intl. pat. Appl. No. PCT/US99/00590 filed Jan. 12, 1999, designatingthe United States of America and published in English, which claims thebenefit of priority of U.S. pat. Appl. No. 60/071,483 filed Jan. 13,1998; the disclosures of such related applications are incorporatedherein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to intranasal immunization with detoxifiedlipooligosaccharide from nontypeable Haemophilus influenzae or Moraxellacatarrhalis.

BACKGROUND OF THE INVENTION

[0003] Nontypeable Haemophilus influenzae (NTHi) is an important causeof otitis media (OM) in children and respiratory tract diseases inadults (Klein, J. O. et al. 1992 Adv Pediatr 39:127-156; Murphy, T. F.et al. 1987 Rev Infect Dis 9:1-15; Musher, D. M. et al. 1983 Ann InternMed 99:344-350). Moraxella (Branhamella) catarrhalis (Catlin, B. W. 1990Clin Microbiol Rev 3:293-320; Doem, G. V. 1986 Diagn Microbiol InfectDis 4:191-201; Enright, M. C., and H. McKenzie 1997 J Med Microbiol46:360-371) is recognized as the third-most-common pathogen causingotitis media and sinusitis in children, after Streptococcus pneumoniaeand nontypeable Haemophilus influenzae (Bluestone, C. D. 1986 Drugs31(Suppl. 3):132-141; Faden, H. et al. 1994 J Infect Dis 169:1312-1317).This gram-negative diplococcus is also a cause of respiratory tractinfections in adults (Boyle, F. M. et al. 1991 Med J Aust 154:592-596;Sarubbi, F. A. et al. 1990 Am J Med 88:9s⁻14S), especially those withchronic obstructive pulmonary diseases (Nicotra, B. et al. 1986 ArchIntern Med 146:890-893) or compromised immune systems (Alaeus, A. and G.Stiernstedt Scand J Infect Dis 23:115-116; Enright, M. C and H.McKenzie. 1997 J Med Microbiol 46:360-371).

[0004] Nontypeable Haemophilus influenzae (NTHi) is an important causeof otitis media in children and of pneumonitis in adults with depressedresistance. Lipooligosaccharide (LOS) is a major surface antigen of NTHiand elicits bactericidal and opsonic antibodies. Gu, X. X. et al. 1996Infect Immun 64:4047-4053 prepared detoxified LOS (dLOS) proteinconjugates from NTHi for use as experimental vaccines. LOS from NTHi9274 was treated with anhydrous hydrazine and had its toxicity reducedto clinically acceptable levels. Hydrazine treatment of NTHi LOSresulted in a 10,000-fold reduction in the level of “endotoxin”, whichis at clinically acceptable levels (W.H.O. Expert Committee onBiological Standardization 1991 W.H.O. Tech Rep Ser 814:15-37) dLOS wasbound to tetanus toxoid (TT) or high-molecular-weight proteins (HMPs)from NTHi through a linker of adipic acid dihydrazide to form dLOS-TT ordLOS-HMP. The molar ratio of the dLOS to protein carriers ranged from26:1 to 50:1. The antigenicity of the conjugates was similar to that ofthe LOS alone as determined by double immunodiffusion. Subcutaneous orintramuscular injection of the conjugates elicited a 28- to 486-foldrise in the level of immunoglobulin G antibodies in mice to thehomologous LOS after two or three injections and a 169- to 243-fold risein the level of immunoglobulin G antibodies in rabbits after twoinjections. The immunogenicity of the conjugates in mice and rabbits wasenhanced by formulation with monophosphoryl lipid A plus trehalosedimycolate. In rabbits, conjugate-induced LOS antibodies inducedcomplement-mediated bactericidal activity against the homologous strain9274 and prototype strain 3189. These results indicate that a detoxifiedLOS-protein conjugate is a candidate vaccine for otitis media andpneumonitis caused by NTHi. Gu, X. X. et al. 1997 Infect Immun65:4488-4493 determined that subcutaneous or intramuscular injections ofdetoxified-lipooligosaccharide (dLOS)-protein conjugates from NTHiprotected against otitis media in chinchillas.

[0005]Moraxella (Branhamella) catarrhalis (M. catarrhalis) is animportant cause of otitis media and sinusitis in children and of lowerrespiratory tract infections in adults. Lipooligosaccharide (LOS) is amajor surface antigen of the bacterium and elicits bactericidalantibodies. Treatment of the LOS from strain ATCC 25238 with anhydroushydrazine reduced its toxicity 20,000-fold, as assayed in the Limulusamebocyte lysate (LAL) test. The detoxified LOS (dLOS) was coupled totetanus toxoid (TT) or high-molecular-weight proteins (HMP) fromnontypeable Haemophilus influenzae through a linker of adipic aciddihydrazide to form dLOS-TT or dLOS-HMP. The molar ratios of dLOS to TTand HMP conjugates were 19:1 and 31:1, respectively. The antigenicity ofthe two conjugates was similar to that of the LOS, as determined bydouble immunodiffusion. Subcutaneous or intramuscular injection of bothconjugates elicited a 50- to 100-fold rise in the geometric mean ofimmunoglobulin G (IgG) to the homologous LOS in mice after threeinjections and a 350- to 700-fold rise of anti-LOS IgG in rabbits aftertwo injections. The immunogenicity of the conjugate was enhanced byformulation with monophosphoryl lipid A plus trehalose dimycolate. Inrabbits, conjugate-induced antisera had complement-mediated bactericidalactivity against the homologous strain and heterologous strains of Mcatarrhalis. These results indicate that a detoxified LOS-proteinconjugate is a candidate for immunization against M catarrhalisdiseases.

[0006] Current pediatric immunization programs include too manyinjections in the first months of life. Oral or nasal vaccine deliveryeliminates the requirement for needles. There is a need for mucosalvaccines against NTHi- and M catarrhalis-caused otitis media in childrenand other NTHi- and M catarrhalis-caused diseases in children andadults.

SUMMARY OF THE INVENTION

[0007] The invention relates to intranasal immunization with detoxifiedlipooligosaccharide from nontypeable Haemophilus influenzae or Moraxellacatarrhalis.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008]FIG. 1 shows the proposed chemical structure of lipid A fromnontypeable Haemophilus influenzae lipooligosaccharide (LOS). R=site ofattachment of the oligosaccharide chain. Hydrazine treatment of LOSremoves primary O-linked fatty acids from 3-hydroxy groups ofdiglucosamine (*) and secondary O-linked fatty acids from hydroxy groupsof 3-hydroxy fatty acids of lipid A (arrow).

[0009]FIG. 2 shows the proposed chemical structure of lipid A fromMoraxella catarrhalis lipooligosaccharide (LOS). R=site of attachment ofthe oligosaccharide chain. Hydrazine treatment of LOS removes primaryO-linked fatty acids from 3-hydroxy groups of diglucosamine (*) andsecondary O-linked fatty acids from hydroxy groups of 3-hydroxy fattyacids of lipid A (arrow).

[0010]FIG. 3. Immunohistochemistry with anti-IgA and anti-IgG stainingin the nose. (A) Anti-IgA (or anti-IgG) staining in control mice, (B)anti-IgA staining in dLOS-TT immunized mice, and (C) anti-IgG stainingin dLOS-TT immunized mice (magnification 400×). Intranasal immunizationwith dLOS-TT dramatically increased the staining with IgA of the mucousblanket, and glandular cells in the nose as compared with the stainingin the control mice. However, staining with anti-IgG was strongly shownonly at the vessels of the nasal tissue in mice immunized with dLOS-TT.The nasal tissue of the control mice was not stained with anti-IgA (oranti-IgG).

[0011]FIG. 4. Bacterial clearance of NTHi strain 9274 from mousenasopharynx. Immunization schedules and mouse grouping were shown inTable 1, footnote a. Mice were challenged with strain 9274 into the nose1 wk after the last immunization and nasal washes were collected at 6 hpost-challenge. Mice immunized with dLOS-TT and CT showed a significantreduction of bacterial recovery by 74% or 76% when compared to those ofthe mice immunized with CT alone or dLOS and CT (*, p<0.05).

[0012]FIG. 5. Binding reactivity of nasal wash (IgA) to homologousstrain and five heterologous strains in whole-cell ELISA. The nasal washfrom mice immunized with dLOS-TT bound strongly to the homologous strain9274 and the heterologous strains 3198, 5657 and 7502 but weakly tostrains 1479 and 2019.

[0013]FIG. 6. Binding reactivity of serum (IgG) to homologous strain andfive heterologous strains in whole-cell ELISA. The observed bindingreactivity was similar to the one observed in nasal wash from miceimmunized with the dLOS-TT (FIG. 5).

[0014]FIG. 7. Silver-stained SDS-PAGE patterns (A) and Western blotanalysis (B and C) of homologous strain and five heterologous strains.Lanes 1 through 6 contain strains 1479, 2019, 3198, 5657, 7502 and 9274.Nasal wash (IgA) from mice immunized with dLOS-TT was reactive stronglyto LOSs from strains 9274, 3198, 5657, and 7502, weakly to 1479 but notto 2019 (B). However, sera (IgG) from mice immunized with the dLOS-TTwere reactive to all LOSs with strong binding in strains 9274, 3198,5657 and 7502 (C). Arrows show each LPS of Ra (upper arrow) and Rc(lower arrow) mutants as markers from Salmonella minnesota.

[0015] FIGS. 8A-C. Specific antibody-forming cells induced by dLOS—CRMconjugate measured by ELISPOT assay. See Table 4, footnote a. (A)IgA-forming cells per million of lymphoid cells; (B) IgG-forming cellsper million of lymphoid cells; (C) IgM-forming cells per million oflymphoid cells. NALT: nasal-associated lymphoid tissue, NP: nasalpassage, CLN: cervical lymph node, PP: Peyer's patch.

[0016] FIGS. 9A-C. Specific antibody-forming cells initiated bydifferent dLOS-protein conjugates. See Table 6, footnote a. NALT:nasal-associated lymphoid tissue, NP: nasal passage, CLN: cervical lymphnode, PP: Peyer's patch.

[0017] FIGS. 10A-B. Comparison of protective effect induced by differentdLOS-protein conjugates in bacterial clearance from mouse nasopharynxand lungs. See Table 6, footnote a. One week after the lastimmunization, mice were challenged with 2×10⁸ CFU of M catarrhalisstrain 25238 per ml in a nebulizer, and nasal washes and lungs werecollected at 6 h postchallenge. The CFU of bacterial recovery from CTgroup compared to that of other group: P<0.01.

[0018]FIG. 11. Comparison of protective effect from differentimmunization regimens in bacterial clearance from mouse nasopharynx. SeeTable 7, footnote a. One week after the last immunization, mice werechallenged with 2×10⁸ CFU of M catarrhalis strain 25238 per ml in anebulizer, and nasal washes were collected at 6 h postchallenge. Lefttwo bars: intranasal immunization, right two bars: subcutaneousinjection.

[0019]FIG. 12. Comparison of protective effect from differentimmunization regimens in bacterial clearance from mouse lungs. See Table7, footnote a. One week after the last immunization, mice werechallenged with 2×10⁸ CFU of M catarrhalis strain 25238 per ml in anebulizer, and lungs were collected at 6 h postchallenge. Left two bars:intranasal immunization, right two bars: subcutaneous injection.

[0020]FIG. 13. Kinetics of bacterial recovery from mouse nasopharynxchallenged with M catarrhalis strain 25238. Mice were intranasallyadministered 4 times at 1-week intervals with 10 μl of PBS containing amixture of 5 μg of dLOS—CRM and 1 μg of CT, or 10 μl of PBS. One weekafter the last immunization, mice were challenged with 5×10⁸ CFU of M.catarrhalis strain 25238 per ml in a nebulizer, and nasal washes orlungs were collected at 0, 3, 6, 12, 24 h postchallenge, respectively.At each time point, immunized mice significantly reduced bacterialrecovery from nasopharynx and lungs, and bacterial recovery becameundetectable within 24 h, postchallenge.

[0021]FIG. 14. Kinetics of bacterial recovery from mouse lungschallenged with M catarrhalis strain 25238. See description of FIG. 13.

DETAILED DESCRIPTION OF THE INVENTION

[0022] The invention relates to an immunogenic composition comprising animmunizing amount of Nontypeable Haemophilus influenzae (NTHi) orMoraxella catarrhalis lipooligosaccharide (LOS) from which at least oneprimary O-linked fatty acid has been removed to form detoxified LOS(dLOS) and an immunogenic carrier covalently linked thereto, optionallywhere the dLOS and the immunogenic carrier are covalently linked by alinker, and a mucosal adjuvant or delivery system.

[0023] In accordance with the present invention, it has now beensurprisingly found that mucosal administration, preferably intranasally,of NTHi or M catarrhalis lipooligosaccharide (LOS) from which at leastone primary O-linked fatty acid has been removed to form detoxified LOS(dLOS) and an immunogenic carrier covalently linked thereto, optionallywhere the dLOS and the immunogenic carrier are covalently linked by alinker, elicits an immunological response and can even inhibitcolonization by NTHi or M catarrhalis and prevent otitis media and otherrespiratory diseases caused by NTHi or M catarrhalis infection.

[0024] Accordingly, in one aspect, the present invention provides amethod for inducing an immunological response in a host, preferably ahuman host, to inhibit colonization by NTHi or M catarrhalis or preventotitis media and other respiratory diseases caused by NTHi or Mcatarrhalis infection by mucosal administration, preferably intranasaladministration, to the host of an effective amount of NTHi or Mcatarrhalis lipooligosaccharide (LOS) from which at least one primaryO-linked fatty acid has been removed to form detoxified LOS (dLOS) andan immunogenic carrier covalently linked thereto, optionally where thedLOS and the immunogenic carrier are covalently linked by a linker, anda mucosal adjuvant or delivery system.

[0025] Moreover, in another aspect, the present invention provides useof an effective amount of NTHi or M catarrhalis lipooligosaccharide(LOS) from which at least one primary O-linked fatty acid has beenremoved to form detoxified LOS (dLOS) and an immunogenic carriercovalently linked thereto, optionally where the dLOS and the immunogeniccarrier are covalently linked by a linker, and a mucosal adjuvant ordelivery system, for mucosal administration, preferably intranasaladministration, to a host, preferably a human host, for inducing animmunological response to inhibit colonization by NTHi or M catarrhalisor prevent otitis media and other respiratory diseases caused by NTHi orM catarrhalis infection.

[0026] The present invention relates to a conjugate vaccine comprisingnontypeable Haemophilus influenzae (NTHi) or Moraxella catarrhalislipooligosaccharide (LOS) from which at least one primary O-linkedesterified fatty acid has been removed to form detoxified LOS (dLOS),and an immunogenic carrier covalently linked thereto, optionally wherethe dLOS and immunogenic carrier are covalently linked by a linker. LOSmay be extracted from NTHi or M catarrhalis and purified according toconventional processes. NTHi and M catarrhalis lipooligosaccharides maybe of any serotype. As a matter of example, serotypes I, II, III, IV andV for NTHi are cited (Campagnari, A. A. et al. 1987 Infect Immun55:882-887; Partick, C. C. et al. 1987 Infect Immun 55:2902-2911), butthe LOS used for the conjugates herein was highly cross-reactive to themajority of NTHi clinical isolates. For M catarrhalis, three major LOSserotypes: A, B and C are cited (Vaneechoutte, M. G. et al. 1990 J ClinMicrobiol 28:182-187). One or several lipooligosaccharides may beconcomitantly administered by the mucosal route. In particular, themedicament, i.e., the vaccine, for mucosal administration may containseveral lipooligosaccharides, each of a particular serotype.

[0027] A proposed chemical structure of lipid A from nontypeableHaemophilus influenzae lipooligosaccharide (LOS) is shown in FIG. 1. Aproposed chemical structure of lipid A from Moraxella catarrhalislipooligosaccharide (LOS) is shown in FIG. 2. The O-linked esterifiedfatty acids shown by the asterisks are defined as primary O-linked fattyacids and those shown by the arrows are defined as secondary O-linkedfatty acids. The conjugate vaccine may also comprise LOS from which bothprimary O-linked fatty acids have been removed. In addition to theremoval of at least one primary O-linked fatty acid from LOS, one orboth of the secondary O-linked fatty acids may also be removed. Thenumber of primary and secondary O-linked fatty acids removed byhydrazine treatment, or by treatment with any other reagent capable ofhydrolyzing these linkages, will depend on the time and temperature ofthe hydrolysis reaction. The determination of the number of fatty acidchains which have been removed during the reaction can be determined bystandard analytical methods including mass spectrometry and nuclearmagnetic resonance (NMR).

[0028] Although the use of hydrazine for detoxification of LOS from NTHior M catarrhalis is described herein, the use of any reagent or enzymecapable of removing at least one primary O-linked fatty acid from LOS iswithin the scope of the present invention. For example, other bases suchas sodium hydroxide, potassium hydroxide, and the like may be used.

[0029] After removal of one or more primary O-linked fatty acids, dLOSis optionally conjugated to a linker, such as adipic acid dihydrazide(ADH), prior to conjugation to an immunogenic carrier protein, such astetanus toxoid (TT). Although ADH is the preferred linker, the use ofany linker capable of stably and efficiently conjugating dLOS to animmunogenic carrier protein is contemplated. The use of linkers is wellknown in the conjugate vaccine field (see Dick et al. ConjugateVaccines, J. M. Cruse and R. E. Lewis, Jr., eds. Karger, New York, pp.48-114, 1989).

[0030] dLOS may be directly covalently bonded to the carrier. This maybe accomplished, for example, by using the cross-linking reagentglutaraldehyde. However, in a preferred embodiment, dLOS and the carrierare separated by a linker. The presence of a linker promotes optimumimmunogenicity of the conjugate and more efficient coupling of the dLOSwith the carrier. Linkers separate the two antigenic components bychains whose length and flexibility can be adjusted as desired. Betweenthe bifunctional sites, the chains can contain a variety of structuralfeatures, including heteroatoms and cleavage sites. Linkers also permitcorresponding increases in translational and rotational characteristicsof the antigens, increasing access of the binding sites to solubleantibodies. Besides ADH, suitable linkers include, for example,heterodifunctional linkers such as ε-aminohexanoic acid, chlorohexanoldimethyl acetal, D-glucuronolactone and p-nitrophenyl amine. Couplingreagents contemplated for use in the present invention includehydroxysuccinimides and carbodiimides. Many other linkers and couplingreagents known to those of ordinary skill in the art are also suitablefor use in the invention (e.g. cystamine). Such compounds are discussedin detail by Dick et al. (Dick et al. Conjugate Vaccines, J. M. Cruseand R. E. Lewis, Jr., eds. Karger, New York, pp. 48-114, 1989).

[0031] The presence of a carrier increases the immunogenicity of thedLOS. Polymeric immunogenic carriers can be a natural or syntheticmaterial containing a primary and/or secondary amino group, an azidogroup or a carboxyl group. The carrier may be water soluble orinsoluble.

[0032] Any one of a variety of immunogenic carrier proteins may be usedin the conjugate vaccine of the present invention. Such classes ofproteins include pili, outer membrane proteins and excreted toxins ofpathogenic bacteria, nontoxic or “toxoid” forms of such excreted toxins,nontoxic proteins antigenically similar to bacterial toxins(cross-reacting materials or CRMs) and other proteins. Nonlimitingexamples of bacterial toxoids contemplated for use in the presentinvention include tetanus toxin/toxoid, diphtheria toxin/toxoid,detoxified P. aeruginosa toxin A, cholera toxin/toxoid, pertussistoxin/toxoid and Clostridium perfringens exotoxins/toxoid. The toxoidforms of these bacterial toxins are preferred. The use of viral proteins(i.e. hepatitis B surface/core antigens; rotavirus VP 7 protein andrespiratory syncytial virus F and G proteins) is also contemplated.

[0033] CRMs include CRM197, antigenically equivalent to diphtheria toxin(Pappenheimer et al. 1972 Immunochem 9:891-906) and CRM3201, agenetically manipulated variant of pertussis toxin (Black et al. 1988Science 240:656-659). The use of immunogenic carrier proteins fromnon-mammalian sources including keyhole limpet hemocyanin, horseshoecrab hemocyanin and plant edestin is also within the scope of theinvention.

[0034] Outer membrane proteins include high molecular weight proteins(HMPs), P4 and P6 from nontypeable Haemophilus influenzae and CD andUSPA from Moraxella catarrhalis. For a list of other outer membraneproteins, see PCT WO98/53851.

[0035] There are many coupling methods which can be envisioned fordLOS-protein conjugates. In the disclosure set forth below, dLOS isselectively activated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC)-mediated ADH derivatization of the terminal3-deoxy-D-manno-2-octulosonic acid (KDO) group of dLOS, followed byEDC-mediated coupling to TT. Alternatively, another method for producingthe instant conjugates involves cystamine derivatization of dLOS, by,for example, EDC-mediated derivatization, followed by disulfideconjugation to N-succimidyl-3-(2-pyridyldithio) propionate-derivatizedprotein. Other methods well known in the art for effecting conjugationof oligosaccharides to immunogenic carrier proteins are also within thescope of the invention. Such methods are described in, for example, U.S.Pat. Nos. 5,153,312 and 5,204,098; and EP 0 497 525; and EP 0 245 045.

[0036] The molar ratio of ADH to dLOS in the reaction mixture istypically between about 10:1 and about 250:1. A molar excess of ADH isused to ensure more efficient coupling and to limit dLOS-dLOS coupling.In a preferred embodiment, the molar ratio is between about 50:1 andabout 150:1; in a most preferred embodiment, the molar ratio is about100:1. Similar ratios of AH-dLOS to both TT and HMP in the reactionmixture are also contemplated. In a preferred embodiment, one ADH perdLOS is present in the AH-dLOS conjugate. In another preferredembodiment, in the final dLOS-carrier protein conjugate, the molar ratioof dLOS to carrier is between about 15 and about 75, preferably betweenabout 25 and about 50.

[0037] Immunogenic compositions including vaccines may be prepared asinhalables, sprays and the like (e.g., nasal spray, aerosol spray orpump spray and the like), e.g., as liquid solutions or emulsions, etc.Aerosol spray preparations can be in a pressurized container with asuitable propellant such as a hydrocarbon propellant. Pump spraydispensers can dispense a metered dose or, a dose having a particularparticle or droplet size. Pump spray dispensers are commerciallyavailable, e.g., from Valois of America, Inc., Connecticut. Nasal spraydispensers are commonly fabricated from a flexible material such asplastic and cause a spray to dispense in response to being squeezed.Anti-inflammatories, such as “Vanceril” are commercially available inoral and nasal aerosol form for mucosal administration; theanti-inflammatory “Vancerase” is commercially available in a pump-spraydispenser for nasal administration; cold remedies such as “Dristan” arecommercially available in nasal spray (squeeze) dispensers (so that thereader is aware that aerosol, pump and squeeze dispensers are known andavailable).

[0038] The lipooligosaccharide may be mixed with pharmaceuticallyacceptable excipients which are compatible therewith. Such excipientsmay include water, saline, dextrose, glycerol, ethanol, and combinationsthereof. The immunogenic compositions and vaccines may further containauxiliary substances, such as wetting or emulsifying agents, pHbuffering agents, or mucosal adjuvants or delivery systems to enhancethe effectiveness thereof.

[0039] For use in the present invention, the lipooligosaccharide iscombined with a mucosal adjuvant or delivery system. See Singh, M. &O'Hagan, D., November 1999 Nature Biotechnology 17:1075-1081; and Ryan,E. J. et al. August 2001 Trends in Biotechnology 19:293-304. Suitablemucosal adjuvants and delivery systems are listed in the table below.TABLE Mucosal Adjuvants and Delivery Systems Aluminum salts ChitosanCytokines (e.g., IL-1, IL-2, IL-12, IFN-γ, GM-CSF) Saponins (e.g., QS21)Muramyl dipeptide (MDP) derivatives CpG oligos Lipopolysaccharide (LPS)of gram-negative bacteria Monophosphoryl Lipid A (MPL) PolyphosphazenesEmulsions (e.g., Freund's, SAF, MF59) Virosomes Iscoms CochleatesPoly(lactide-co-glycolides) (PLG) microparticles Poloxamer particlesVirus-like particles Heat-labile enterotoxin (LT), LT B subunit Choleratoxin (CT), CT B subunit Mutant toxins (e.g., LTK63 and LTR72)Microparticles Liposomes

[0040] The mucosal administration preferably is effected intranasally,e.g., to the olfactory mucosa, to provide protection to the host againstboth bacterial colonization and systemic infection. The intranasaladministration also may provide protection to the host against pulmonaryinfection as well as protection to the host against an infectionstarting as a pulmonary infection. However, the mucosal administrationcan also involve respiratory mucosa, gingival mucosa or alveolar mucosa.Thus, the administration can be perlingual or sublingual or into themouth or respiratory tract; but intranasal administration is preferred.

[0041] Compositions of the invention, especially for nasaladministration, are conveniently provided as isotonic aqueous solutions,suspensions or viscous compositions which may be buffered to a selectedpH. The viscous compositions may be in the form of gels, lotions,ointments, creams and the like and will typically contain a sufficientamount of a thickening agent so that the viscosity is from about 2500 to6500 cps, although more viscous compositions, even up to 10,000 cps maybe employed. Viscous compositions have a viscosity preferably of 2500 to5000 cps, since above that range they become more difficult toadminister.

[0042] Liquid sprays and drops are normally easier to prepare than gelsand other viscous compositions. Additionally, they are somewhat moreconvenient to administer, especially in multi-dose situations. Viscouscompositions, on the other hand can be formulated within the appropriateviscosity range to provide longer contact periods with mucosa, such asthe nasal mucosa.

[0043] Suitable nontoxic pharmaceutically acceptable carriers, andespecially nasal carriers, will be apparent to those skilled in the artof pharmaceutical and especially nasal pharmaceutical formulations. Forthose not skilled in the art, reference is made to the text entitledRemington 's Pharmaceutical Sciences, a reference book in the field.Obviously, the choice of suitable carriers will depend on the exactnature of the particular mucosal dosage form, e.g., nasal dosage form,required [e.g., whether the composition is to be formulated into asolution such as a nasal solution (for use as drops or as a spray), anasal suspension, a nasal ointment, a nasal gel or another nasal form].Preferred mucosal and especially nasal dosage forms are solutions,suspensions and gels, which normally contain a major amount of water(preferably purified water) in addition to the antigen (PspA). Minoramounts of other ingredients such as pH adjusters (e.g., a base such asNaOH), emulsifiers or dispersing agents, buffering agents,preservatives, wetting agents and jelling agents (e.g., methylcellulose)may also be present. The mucosal (especially nasal) compositions can beisotonic, i.e., it can have the same osmotic pressure as blood andlacrimal fluid.

[0044] The desired isotonicity of the compositions of this invention maybe accomplished using sodium chloride, or other pharmaceuticallyacceptable agents such as dextrose, boric acid, sodium tartrate,propylene glycol or other inorganic or organic solutes. Sodium chlorideis preferred particularly for buffers containing sodium ions.

[0045] Viscosity of the compositions may be maintained at the selectedlevel using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose, hydroxyproplcellulose, carbomer, and the like. The preferred concentration of thethickener will depend upon the agent selected. The important point is touse an amount which will achieve the selected viscosity. Viscouscompositions are normally prepared from solutions by the addition ofsuch thickening agents.

[0046] Compositions within the scope of this invention can contain ahumectant to inhibit drying of the mucous membrane and to preventirritation. Any of a variety of pharmaceutically acceptable humectantscan be employed including, for example sorbitol, propylene glycol orglycerol. As with the thickeners, the concentration will vary with theselected agent, although the presence or absence of these agents, ortheir concentration, is not an essential feature of the invention.

[0047] Enhanced absorption across the mucosal and especially nasalmembrane can be accomplished employing a pharmaceutically acceptablesurfactant. Typically useful surfactants for compositions includepolyoxyethylene derivatives of fatty acid partial esters of sorbitolanhydrides such as Tween 80, Polyoxyl 40 Stearate, Polyoxyethylene 50Stearate and Octoxynol. The usual concentration is form 1% to 10% basedon the total weight.

[0048] A pharmaceutically acceptable preservative can be employed toincrease the shelf-life of the compositions. Benzyl alcohol may besuitable, although a variety of preservatives including, for example,Parabens, thimerosal, chlorobutanol, or bezalkonium chloride may also beemployed. A suitable concentration of the preservative will be from0.02% to 2% based on the total weight although there may be appreciablevariation depending upon the agent selected.

[0049] Those skilled in the art will recognize that the components ofthe compositions must be selected to be chemically inert with respect tothe lipooliogosaccharide. This will present no problem to those skilledin chemical and pharmaceutical principles, or problems can be readilyavoided by reference to standard texts or by simple experiments (notinvolving undue experimentation), from this disclosure.

[0050] The therapeutically effective compositions of this invention areprepared by mixing the ingredients following generally acceptedprocedures. For example the selected components may be simply mixed in ablender, or other standard device to produce a concentrated mixturewhich may then be adjusted to the final concentration and viscosity bythe addition of water or thickening agent and possibly a buffer tocontrol pH or an additional solute to control tonicity. Generally the pHmay be from about 3 to 7.5. Compositions can be administered in dosagesand by techniques well known to those skilled in the medical arts takinginto consideration such factors as the age, sex, weight, and conditionof the particular patient, and the mucosal route of administration.Dosages for humans or other mammals can be determined without undueexperimentation by the skilled artisan from experiments involving mice,rabbits, chinchillas, etc.

[0051] The vaccine composition which is administered intranasally asprovided herein may be formulated in any convenient manner and in adosage formulation consistent with the mode of administration and theelicitation of a protective response. The quantity of antigen to beadministered depends on the subject to be immunized and the form of theantigen. Precise amounts and form of the antigen to be administereddepend on the judgement of the practitioner. However, suitable dosageranges are readily determinable by those skilled in the art and may beof the order of micrograms to milligrams. Suitable regimes for initialadministration and booster doses also are variable, but may include aninitial administration followed by subsequent administrations.

[0052] In summary, the lipooligosaccharides may conventionally be usedin the preparation of the medicament e.g., vaccine. In particular, thelipooligosaccharides may be formulated with a diluent or apharmaceutically acceptable carrier e.g., a buffer or a saline. Thevaccine may additionally contain usual ingredients such as a stabilizeror as already mentioned above, a mucosal adjuvant or delivery system. Ina general manner, these products are selected according to standardpharmaceutical practices as described in Remington 's PharmaceuticalSciences, a reference book in the field.

[0053] In a vaccination protocol, the vaccine may be administered by themucosal route, as a unique dose or preferably, several times e.g.,twice, three or four times at week or month intervals, according to aprime/boost mode. The appropriate dosage depends upon variousparameters, including the number of valencies contained in the vaccine,the serotypes of the lipooligosaccharides and the age of the recipient.It is indicated that a vaccine dose suitably contain per valency, from0.5 to 100 μg, preferably from 1 to 50 μg, more preferably from 1 to 10μg of lipooligosaccharide. A dose is advantageously under a volume offrom 0.1 to 2 ml.

[0054] The vaccination protocol may be a strict mucosal protocol or amix protocol in which the priming dose of the vaccine is administered bythe mucosal e.g., intranasal route and the boosting dose(s) is (are)parenterally administered or vice versa.

Intranasal Immunization with Lipooligosaccharide-Based Conjugate Vaccinefrom Nontypeable Haemophilus influenzae Inhibits Bacterial Colonizationin Mouse Nasopharynx.

[0055] Previous studies reported as Gu, X. X. et al. 1996 Infect Immun64:4047-4053 and Gu, X. X. et al. 1997 Infect Immun 65:4488-4493demonstrated that systemic immunization with detoxifiedlipooligosaccharide (LOS) conjugate vaccines from nontypeableHaemophilus influenzae (NTHi) elicited LOS-specific antibodies in miceand rabbits and resulted in protection against experimental otitis mediain chinchillas. In this disclosure, we investigated if intranasalimmunization with such a detoxified LOS-tetanus toxoid (dLOS-TT) vaccinewould generate protective immunity against NTHi in a mouse model ofnasopharyngeal colonization. The results demonstrated that intranasalimmunization with dLOS-TT plus adjuvant cholera toxin (CT) significantlyinduced LOS-specific IgA antibodies in mouse external secretions,especially in nasal wash (90-fold) followed by bronchoalveolar lavagefluid (25-fold), saliva (13-fold) and fecal extract (3-fold).LOS-specific IgA antibody forming cells were also found in mucosal andlymphoid tissues with the highest number in nasal passage (528 per 10⁶cells). In addition, the intranasal immunization elicited a significantrise of LOS-specific IgG (32-fold) and IgA (13-fold) in serum. Whenthese immunized mice were challenged through the nose with 10⁷ livebacteria of strain 9274, the vaccine group showed a significantreduction of NTHi by 74% and 76%, compared to that of control groupswith CT alone or dLOS plus CT (p<0.05). Negative correlations were foundbetween bacterial counts and the levels of nasal wash IgA or IgG, salivaIgA or serum IgG. The clearance of five heterologous strains wereinvestigated and revealed a significant clearance in strains 3198, 5657and 7502 but not in strains 1479 and 2019. These data indicate thatintranasal immunization with dLOS-TT vaccine elicits both mucosal andsystemic immunity against NTHi colonization in a mouse model ofnasopharyngeal colonization. Therefore, it is envisioned as a usefulstrategy in humans to inhibit NTHi colonization and prevent otitis mediaand other respiratory diseases caused by NTHi infection.

[0056] Animals. Female BALB/c mice (6 weeks) were purchased from Taconicfarms Inc. (Germantown, N.Y.). The mice were in an animal facility inaccordance with National Institutes of Health guidelines under animalstudy protocol 1009-01.

[0057] NTHi LOS and conjugate vaccine. NTHi strain 9274 and fiveprototype strains 1479, 2019, 3198, 5657 and 7502 were obtained from M.A. Apicella, University of Iowa (Campagnari, A. A. et al. 1987 InfectImmun 55:882-887). LOS of NTHi strain 9274 was extracted from cells byhot phenol water, and then purified by gel filtration as describedpreviously (Gu, X. X. et al. 1995 Infect Immun 63:4115-4120). Proteincontent was about 1% and nucleic acid content was less than 1%.Detoxification of the LOS, conjugation of dLOS to TT, andcharacterization of dLOS-TT from strain 9274 were described previously(Gu, X. X. et al. 1996 Infect Immun 64:4047-4053). The composition ofdLOS-TT was 638 μg of dLOS and 901 μg of TT per ml with a molar ratio ofdLOS to TT at 35:1.

[0058] Bacterial growth and LOS puriflcation. NTHi 9274, isolated frommiddle ear fluid removed from a patient with OM, was provided by M. A.Apicella, University of Iowa. The strain was grown on chocolate agar at37° C. under 5% CO₂ for 8 h and transferred to 200 ml of 3% brain heartinfusion medium (Difco Laboratories, Detroit, Mich.) containing NAD (5μg/ml) and hemin (2 μg/ml) (Sigma Chemical Co., St. Louis, Mo.) in a500-ml bottle. The bottle was incubated at 150 rpm in an incubatorshaker (model G-25; New Brunswick Scientific, Co. Edison, N.J.) at 37°C. overnight. The culture was transferred to five 2.8-liter baffledFembach flasks, each of which contained 1.4 liters of the same medium.The flasks were shaken at 140 rpm and maintained at 37° C. for 24 h. Theculture was centrifuged at 15,000×g at 4° C. for 30 min to separate thecells and the supernatant. LOS was purified from cells by a modifiedphenol-water extraction (Gu, X. X. et al. 1995 Infect Immun63:4115-4120) and from the culture supernatant by gel filtration (Gu, X.X. and Tsai, C. M. 1993 Anal Biochem 196:311-318). The protein andnucleic acid contents of both purified LOSs were less than 1% (Smith, P.K. et al. 1985 Anal Biochem 150:76-85; Warburg, O. and W. Christian 1942Biochem Z 310:385-421).

[0059] Detoxification of LOS. Anhydrous hydrazine treatment oflipopolysaccharides (LPSS) under mild condition removes esterified fattyacids from lipid A (Gupta, R. K. et al. 1992 Infect Immun 60:3201-3208).LOS (160 mg), each lot, was dried over P₂O₅ for 3 days, suspended in 16ml of anhydrous hydrazine (Sigma), and incubated at 37° C. for 2 h withmixing every 15 min. This suspension was cooled on ice and addeddropwise to cold acetone in an ice bath until a precipitate formed (>90%acetone). The mixture was centrifuged at 5,000×g at 5° C. for 30 min.The pellet was washed twice with cold acetone and dissolved inpyrogen-free water at a final concentration of 20 mg/ml. The reactionmixture was ultracentrifuged at 150,000×g at 5° C. for 3 h. Thesupernatant was freeze-dried and passed through a column (1.6 by 90 cm)of Sephadex G-50 (Pharmacia LKB Biotechnology, Uppsala, Sweden), elutedwith 25 mM ammonium acetate, and monitored with a differentialrefractometer (R-400; Waters, Milford, Mass.). The eluate was assayedfor carbohydrate by the phenol-sulfuric acid method (Dubois, M. et al.1956 Anal Biochem 28:250-256). The carbohydrate-containing fractionswere pooled, freeze-dried three times to remove the salt, and designateddLOS. The yields of the dLOS from three lots ranged from 48 to 55% byweight. For all material and reagent preparations, glassware was bakedand pyrogen-free water was used.

[0060] Derivatization or dLOS. Adipic acid dihydrazide (ADH) (AldrichChemical Co., Milwaukee, Wis.) was bound to the carboxyl group of theKDO moiety of the dLOS to form adipic hydrazide (AH)-dLOS derivativeswith 1-ethy-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) andN-hydroxysulfosuccinimide (Pierce) (Gu, X. X. and C. M. Tsai 1993 InfectImmun 61:1873-1880; Staros, J. V. et al. 1986 Anal Biochem 156:220-222).dLOS (70 mg) was dissolved in 7 ml of 345 mM ADH (the molar ratio of ADHto LOS is ˜100:1 based on an estimated 3,000 M_(r) for dLOS) (Gibson, B.W. et al. 1993 J Bacteriol 175:2702-2712; Helander, J. M. et al. 1988Eur J Biochem 177:483-492). N-Hydroxysulfosuccinimide was added to aconcentration of 8 mM, the pH was adjusted to 4.8 with 1 M HCl, and EDCwas added to a concentration of 0.1 M. The reaction mixture was stirredand maintained at pH 4.8+0.2 with 1 M HCl for 3 h at room temperature.It was adjusted to pH 7.0 with NaOH and passed through the G-50 columnas described above. The eluate was assayed for carbohydrate and for AHby a modification of a previously described method (Kemp, A. H. and M.R. A. Morgan 1986 J Immunol Methods 94:65-72) by measuring the A₄₉₀ ofAH groups. The peaks containing both carbohydrate and AH were pooled,freeze-dried three times to remove the salt, and designated AH-dLOS.AH-dLOS was measured for its composition with dLOS and ADH as standards(Dubois, M. et al. 1956 Anal Biochem 28:250-256; Kemp, A. H. and M. R.A. Morgan 1986 J Immunol Methods 94:65-72).

[0061] Conjugation of AH-dLOS to proteins. TT was obtained fromConnaught Laboratories, Inc., Swiftwater, Pa. HMP was purified from NTHi12 (Barenkamp, S. J. 1996 Infect Immun 64:1246-¹²⁵I). AH-dLOS wascoupled to carboxyl groups on TT or HMP at pH 5.6 with EDC. AH-dLOS (20mg) was dissolved in 2 ml of water and mixed with 10 mg of TT (5.9mg/ml) or with 8 mg of HMP (4 mg/ml). The molar ratio of AH-dLOS to bothTT (M_(r) 150,000) and HMP (M_(r) 120,000) was ˜100:1. The pH wasadjusted to 5.6 with 0.1 M HCl, and EDC was added to a concentration of0.1 M. The reaction mixture was stirred for 1 to 3 h at roomtemperature; the pH was maintained at 5.6+0.2 with 0.1 M HCl. Thereaction mixture was adjusted to pH 7.0, centrifuged at 1,000×g for 10min, and passed through a column (1.6 by 90 cm) of Sephacryl S-300 in0.9% NaCl. Peaks that contained both protein and carbohydrate werepooled and designated dLOS-TT or dLOS-HMP. Both conjugates were analyzedfor their composition of carbohydrate and protein with dLOS and bovineserum albumin (BSA) as standards (Dubois, M. et al. 1956 Anal Biochem28:250-256; Smith, P. K. et al. 1985 Anal Biochem 150:76-85).

[0062] Immunization and sample collection. Mice were immunized nasallywith 10 μl of phosphate-buffered saline (PBS) containing a mixture of 5μg of dLOS-TT and 1 μg of cholera toxin (List Biological Laboratories,Campbell, Calif.) as an adjuvant. Control mice intranasally received 10μl of PBS containing 5 μg of dLOS and/or 1 μg of CT. Each dose waspipetted into the mouse nostril (5 μl each side) under anesthesia withintraperitoneal injection of 0.1 ml of 2% ketamine and 0.2% xylazine.Immunizations were given 5 times on days 0, 7, 14, 21 and 28. On day 35,one set of mice was used for bacterial challenge while another set wasused for sample collections only described as follows. Nasal washes,saliva, bronchoalveolar lavage fluids (BALFs), fecal extracts, and serawere collected from mice of each group under anesthesia as describedbefore (Kurono, Y. et al. 1999 J Infect Dis 180:122-132). Briefly,salivary samples were obtained following intraperitoneal injection with0.1 ml of 0.1% pilocarpine (Sigma, St. Louis, Mo.) in PBS to inducesalivary secretion. Blood samples were collected from axillary artery.After removal of the mandible, the nasal cavity was gently flushed fromposterior opening of the nose with 200 μl of PBS and nasal washes werecollected from the anterior openings of the nose. BALF was obtained byirrigation with 1 ml of PBS through a blunted needle inserted into thetrachea after incision. Fecal extract samples were obtained by addingweighed pellets to PBS containing 0.01% sodium azide (100 mg of fecalsamples/ml) according to the method of deVos and Dick (Gu, X. X. et al.1996 Infect Immun 64:4047-4053). Blood and fecal samples werecentrifuged, and the supernatants were collected.

[0063] Preparation of single cell suspension. On day 35, nasal passages,nasal-associated lymphoid tissues (NALTs), spleens, cervical lymph nodes(CLNs), lungs, small intestines and submandibular glands (SMGs) werecollected from mice. Single cell suspensions were prepared from nasalpassages, NALTs, spleens, CLNs, lungs and SMGs by a gentle teasingthrough stainless steel mesh (Asanuma, H. et al. 1997 J Immunol Methods202:123-131). Small intestines were dissociated with 0.5 mg/mlcollagenase Type IV (Sigma) to obtain single-cell suspensions afterremoval of Peyer's patches. Each single-cell suspension sample exceptfor NALTs, spleens and CLNs was centrifuged over a discontinuous Percollgradient (Pharmacia, Uppsala, Sweden), and mononuclear cells (MNCs) atthe interface of the 40% and 75% layers were collected. Then, MNCs weresuspended in complete medium (1 liter of RPMI1640 supplemented with 1%of nonessential amino acid solution, 1 mM HEPES, 100,000 U ofpenicillin, 100 μg of streptomycin, 40 mg of gentamicin, and 10% fetalcalf serum). The number and viability of MNCs were examined by trypanblue dye exclusion.

[0064] Detection of LOS-specific antibodies by ELISA. Specific anti-LOSantibodies in nasal wash, saliva and serum were determined by ELISA withstrain 9274 LOS as coating antigen (10 μg/ml) (Gu, X. X. et al. 1996Infect Immun 64:4047-4053). Samples of naive mice were served asnegative controls. The negative controls gave optical density readingsof less than 0.1 for IgA, IgG and IgM in serum, and 0.01 in externalsecretions. The antibody endpoint titer was defined as the highestdilution of samples giving an optical density two-fold greater than thatof the negative controls at 30 min.

[0065] Detection of LOS-specific antibody-forming cells (AFCs) byenzyme-linked immunospot (ELISPOT) assay. For the enumeration ofLOS-specific immunoglobulin-producing cells, the numbers of LOS-specificIgA-, IgG-, and IgM-producing cells in NALT, NP, SMG, spleen, CLN, lung,and small intestine were determined with ELISPOT assay (Kodama, S. etal. 2000 Infect Immun 68:2294-2300. Briefly, 96-well filtration plateswith a nitrocellulose base (Millititer HA; Millipore Corp., Bedford,Mass.) were coated with 100 μl of strain 9274 LOS (10 μg/ml) andincubated overnight at 4° C. The plates were washed three times with PBSand then blocked with complete medium for 1 h. After removing theblocking medium, test cells in complete medium were added at variousconcentrations and cultured at 37° C. with 5% CO₂ for 6 h. After theincubation, the plates were washed thoroughly with PBS and then with PBScontaining 0.05% Tween 20 (PBS-T). For capture of secreting antibodies,biotinylated goat anti-mouse IgA, IgG, or IgM (Sigma) was added in PBS-Tat 1:1,000. After overnight incubation at 4° C., the plates were washedfive times with PBS-T, and incubated with 5 μg/ml of avidin-peroxidaseconjugates (Sigma) in PBS-T for 1 h at room temperature. After washingwith PBS-T and PBS three times for each, spots were developed in4-chloro-1-naphthol solution for 10 min. The reaction was stopped bywashing with water. The plate were dried and dark blue-purple coloredspots were counted as LOS-specific AFCs under a stereo microscope.

[0066] Immunohistochemistry for IgA-, IgG-, IgM-positive cells in thenose. For histological observation, the mice were euthanized on day 35and then perfused transcardially with PBS, followed by perfusion with10% neutral buffered formalin. Mouse heads were removed and fixed in 10%formalin for 6 hr and decalcified with 0.12 M ethylenediaminetetraacetic acid (EDTA, pH 7.0) for 2 weeks. After dehydration, thetissues were embedded in paraffin. For detection of IgA, IgG,IgM-positive cells in the nose, vertical-serial section (6 μm thickness)were prepared. Specimens were dehydrated through a graded series ofethanol and treated with 3% hydrogen peroxide in absolute methanol for30 min. Sections were exposed to 5% normal goat serum in PBS for 30 minand then incubated overnight with biotinylated goat anti-mouse IgA, IgG,or IgM in 1% bovine serum albumin (BSA)-PBS. After rinsing with PBS,sections were incubated with avidin-biotin complex (Vector Laboratories,Burlingame, Calif.) for 1 h and developed in 0.05%3,3′-diaminobenzidine-0.01% H₂O₂ substrate medium in 0.1M PBS for 8 min.

[0067] Bacterial challenge in nasopharynx. To examine the effect of thedLOS-TT vaccine on NTHi clearance in nasopharynx, the mice immunizedwith different antigens were challenged with the homologous strain 9274.The strain was grown on chocolate agar at 37° C. under 5% CO₂ for 16 h,and then 3-5 clones were transferred to another plate and incubated for4 h. A bacterial suspension was prepared to the concentration of 4˜6×10⁶CFU/ml in PBS and stored on ice until use. The bacterial concentrationwas determined by a 65% transmission at wavelength 540 nm, and confirmedby counting the colonies after overnight incubation. The mice wereintranasally inoculated with 10 μl of the bacterial suspension on day35. Six hours postchallenge, nasal washes were collected and dilutedserially in PBS, and 50 μl of the diluted samples were plated onchocolate agar. Bacterial colonies were counted after overnightincubation. To investigate correlation between antibody levels andbacterial clearance of strain 9274, saliva, BALF, fecal extract andserum samples were collected from each mouse simultaneously. To examinethe effect of the vaccine on heterologous NTHi, strains 1479, 2019,3198, 5657 and 7502 were used based on the same procedure except onlyone control group (CT) was included since no significant difference wasfound between control groups.

[0068] Whole cell ELISA. To examine the cross-reactivity of antibodiesin nasal wash (IgA) and sera (IgG) elicited by the vaccine againstheterologous NTHi strains, the homologous strain 9274 and strains 1479,2019, 3198, 5657 and 7502 were suspended in PBS to an optical density of65% transmission at 540 nm. Microtiter plates were coated with 100 μl ofthe suspension and evaporated at 37° C. Other steps were the same asdescribed for the LOS ELISA except 3% of BSA-PBS was used for blockingand 1:15 dilution used for nasal wash or serum samples.

[0069] Western blot analysis. For characterization of antibodies inexternal secretions and sera, Western blot analysis was performed withthe homologous strain 9274 and five heterologous strains. Each bacterialsuspension was adjusted to a protein concentration of 2 mg/ml. Thesuspensions were boiled at 100° C. for 10 min in digestion buffer,subjected to SDS-PAGE in a 15% polyacrylamide gel and then transferredonto nitrocellulose membranes at 250 mA for 6 h (Gu, X. X. et al. 1992 JClin Microbiol 30:2047-2053). After blocking with 3% BSA-Tris bufferedsaline (TBS) for 1 h, the membranes were incubated with nasal wash orserum sample (1:10) for 3 h, followed by biotinylated goat anti-mouseIgA or IgG for 2 h. The membranes were washed with TBS-T, and incubatedwith avidin-peroxidase conjugate for 1 h. After washing with TBS, themembranes were developed with 4-chloro-1-naphthol solution. A duplicategel was silver-stained after SDS-PAGE.

[0070] Statistical analysis. Antibody levels were expressed as thegeometric mean (GM) ELISA titers (reciprocal) of n independentobservations (±SD range). AFCs were expressed as a mean of n independentobservations (±SD). Bacterial concentration was expressed as GM CFU of nindependent observations (±SD). Differences between vaccine and controlgroups were determined using Student's t-test and P values smaller than0.05 were considered significant. Correlation between bacterialconcentration and IgA or IgG titer was analyzed by Pearson's productmoment method (null hypothesis: Ho: P=0; alternative hypothesis: H₁:P<0, significantly).

RESULTS

[0071] TABLE 1 Murine antibody responses to NTHi 9274 LOS elicited bydLOS-TT conjugate GM antibody ELISA titers (±SD range)^(b) Immunogen^(a)Isotype Saliva Nasal wash BALF^(c) Fecal Extract Serum DLOS-TT + CT IgA63 (28-140)** 452 (205-990)** 128 (24-692)** 16 (6-42)** 125 (61-257)**IgG 13 (4-38)** 16 (8-31)** 25 (8-27)** 6 (4-8) 320 (131-780)** IgM 5(5) 5 (4-7) 6 (4-11) 5 (4-7) 10 (10) DLOS + CT IgA 6 (4-9) 7 (4-12) 6(4-9) 5 (5) 12 (8-20) IgG 5 (5) 6 (4-8) 6 (4-9) 5 (5) 10 (10) IgM 5 (5)5 (5) 5 (5) 5 (5) 10 (10) CT IgA 5 (5) 5 (5) 5 (5) 5 (5) 10 (10) IgG 5(5) 5 (5) 5 (5) 5 (5) 10 (10) IgM 5 (5) 5 (5) 5 (5) 5 (5) 10 (10)

[0072] TABLE 2 LOS-specific AFCs in mucosal and lymphoid tissueselicited by dLOS-TT conjugate LOS-specific AFCs/10⁶ MNCs (mean ± SD)^(b)Nasal Immunogen^(a) Isotype NALT^(c) Passage SMG^(c) Lung IntestineCLN^(c) Spleen dLOS-TT + CT IgA 27 ± 8 528 ± 40  9 ± 1 12 ± 2 6 ± 1  9 ±1 4 ± 1 IgG 0 9 ± 6 0 0 0 10 ± 8 3 ± 2 IgM 0 0 0 0 0 0 0 DLOS + CT IgA 02 ± 2 0 0 0 0 0 IgG 0 0 0 0 0 0 0 IgM 0 0 0 0 0 0 0 CT IgA 0 0 0 0 0 0 0IgG 0 0 0 0 0 0 0 IgM 0 0 0 0 0 0 0

[0073] TABLE 3 Effect of intranasal immunization with dLOS-TT conjugateon bacterial clearance of heterologous NTHi strains from mousenasopharynx Strain Bacterial recovery GM Bacterial concentration^(a)(±SD range) reduction (cfu/ml) Immunogen^(b) (cfu/ml) (%)^(c) 1479dLOS-TT + CT 1324 (36-5676)  50%  (5 × 10⁹) CT 2643 (841-8303) 2019dLOS-TT + CT 2870 (935-8807) 29%  (6 × 10⁹) CT  4054 (1369-12006) 3198dLOS-TT + CT  3347 (1259-8902) 65%* (4 × 10⁹) CT  9727 (3336-28365) 5657DLOS-TT + CT  780 (340-1792) 63%* (5 × 10⁹) CT 2041 (531-7998) 7502DLOS-TT + CT 2050 (726-5793) 57%* (6 × 10⁹) CT  4788 (1779-12835)

[0074] LOS-specific immune responses in external secretions and serumsamples. LOS-specific immune responses were elicited significantly byintranasal immunization with dLOS-TT and CT but not controls (Table 1).LOS-specific IgA titers in external secretions and in serum wereincreased by dLOS-TT and CT, especially in nasal wash (90-fold), BALF(26-fold), saliva (13-fold) and serum (13-fold), whereas slight increaseof LOS-specific IgA in fecal extract was found (3-fold) when compared tothat of CT controls. LOS-specific IgG titers in serum were increasedsignificantly with dLOS-TT and CT by 32-fold, while LOS-specific IgGantibodies in external secretions except for fecal extract were alsoelevated by 3 to 5-fold when compared to that of CT controls. NoLOS-specific IgM was detected and no difference of antibody titers foundbetween two control groups: dLOS plus CT and CT alone (p>0.05).

[0075] LOS-specific antibody-forming cells (AFCs) in mucosal effectortissues. Intranasal immunization with dLOS-TT and CT resulted indetection of LOS-specific IgA AFCs in all tissues tested, includingdistant organs such as intestine and spleen (Table 2). The majority ofLOS-specific IgA AFCs were located in nasal passage (528 per 10⁶ cells),followed by a small amount in other tested tissues. The dominant isotypeof LOS-specific AFCs was IgA, followed by small numbers of IgG but notIgM. LOS-specific IgG AFCs were only detected in nasal passage, CLN andspleen. Intranasal immunization with dLOS and CT elicited 2 LOS-specificIgA AFCs in nasal passage but not in other tested tissues. No AFC wasfound in any tissues from mice immunized with CT.

[0076] Immunohistochemical staining of the nose. Immunohistochemicalstaining of noses with anti-IgA (FIG. 3) revealed that the mouseimmunized with the dLOS-TT vaccine showed positive staining in themucous blanket and glandular tissues (B) as compared with the controlmouse (A). A large number of IgA-positive cells were found in nasalsubepithelial layer and nasal glands. In contrast, staining withanti-IgG in the mouse immunized with the dLOS-TT vaccine was only seenin the area of the vessels but not the glandular tissue (C). The nasalmucosa of the control mice was not stained with anti-IgG, and bothvaccine-immunized and control mice showed no staining with anti-IgM inthe nose.

[0077] Bacterial clearance from nasopharynx. Since intranasalimmunization with dLOS-TT vaccine induced high levels of LOS-specificIgA antibodies in nasal wash and IgG antibodies in serum, it wasimportant to examine whether the NTHi LOS specific immune responsescontributed to the clearance of NTHi colonization in the nasal tract.Bacterial colonization of the homologous strain inoculated into themouse nasopharynx is shown in FIG. 4. The mice immunized with dLOS-TTand CT showed a significant reduction of bacterial recovery by 74% or76% when compared to those of the mice immunized with CT alone or dLOSand CT (p<0.05). Relationship between LOS-specific antibody titers andbacterial counts from nasopharynx was further analyzed in nasal wash,saliva, BALF, fecal extract and serum from dLOS-TT and CT immunized andCT immunized mice. Negative correlation with bacteria was found in nasalwash IgA (r=−0.56, p=0.0085) or IgG (r=−0.63, p=0.0025), saliva IgA(r=−0.45, p=0.0447), or serum IgG (r=−0.65, p=0.014).

[0078] Heterologous bacterial clearance from nasopharynx. Since strain9274 LOS contains common LOS epitopes, bacterial clearance ofheterologous strains was performed in mice immunized with or withoutdLOS-TT in CT (Table 3). Significant inhibition in bacterialcolonization was seen in 3 out of 5 strains (3198, 5657 and 7502) with areduction of 57 to 65%, when compared to the mice immunized with CTalone (p<0.05).

[0079] Cross-reactivity of LOS antibodies with heterologous strains. Thecross-reactivity of antibodies elicited by NTHi 9274 dLOS-TT and CTagainst heterologous strains was analyzed by whole cell ELISA with bothnasal wash (mainly IgA) and sera (mainly IgG) (FIGS. 5 and 6). Nasalwash IgA bound strongly to not only the homologous strain but also theheterologous strains 3198, 5657, and 7502 when compared with thecontrols. Binding reactivity of serum IgG also showed the same tendencyas the nasal wash IgA. However, bindings to the heterologous strains1479 and 2019 were weak in both nasal wash and serum antibodies. Thecontrol mice showed low background binding in nasal wash and mediumbackground in serum to all strains. Both nasal wash and serum sampleswere further tested in Western blot with all above strains (FIG. 7).Nasal wash IgA from mice immunized with dLOS-TT and CT was reactivestrongly to LOSs of strains 9274 and 3198, moderately to 5657 and 7502,and weakly to 1479 LOS but not 2019 LOS. However, the serum IgG wasreactive to all with a strong binding to LOSs of strains 9274 and 3198.

[0080] Conclusions. Intranasal immunization with a NTHi dLOS-TTconjugate vaccine elicited LOS-specific IgA antibodies in local anddistant external secretions as well as LOS-specific IgA AFCs in mucosaleffector tissues (nasal passage, SMG, lung and intestine) and lymphoidtissues (NALT, CLN and spleen). It also generated significantLOS-specific IgG antibodies in serum. This is the first demonstration atintranasal administration of a LOS-based conjugate elicitingantigen-specific mucosal and systemic immune responses although severalrecent studies have shown similar results by capsular polysaccharideconjugates from Streptococcal pneumoniae, group B Streptococci orHaemophilus influenzae type b (Bergquist, C., T. Lagergard, and J.Holmgren 1998 Apmis 106:800-806; Jakobsen, H. et al. 1999 Infect Immun67:4128-4133; Jakobsen, H. et al. 1999 Infect Immun 67:5892-5897; Shen,X. et al. 2000 Infect Immun 68:5749-5755). In summary, intranasalimmunization with a LOS-based conjugate vaccine elicited LOS-specificmucosal and systemic immunity, which inhibited not only the homologousbut also the heterologous bacterial adherence in a mouse model ofnasopharyngeal colonization. Therefore, it is envisioned as beingeffective in humans with an appropriate mucosal adjuvant or deliverysystem to inhibit NTHi colonization and prevent otitis media and otherrespiratory diseases caused by NTHi infection.

Intranasal Immunization with Detoxified Lipooligosaccharides fromMoraxella catarrhalis Conjugated to a Protein Elicits Protection in aMouse Model of Colonization.

[0081]Moraxella catarrhalis is a significant cause of otitis media inchildren. Lipooligosaccharide (LOS) is a major surface antigen of Mcatarrhalis and a potential vaccine candidate. In order to address themucosal immunity of detoxified LOS (dLOS)-protein conjugate vaccines andtheir potential roles on mucosal surfaces, BALB/c mice were immunizedintranasally with a mixture of dLOS—CRM (the diphtheria toxincross-reactive mutant protein) and cholera toxin (CT) as an adjuvant,dLOS plus CT, or CT only. After immunization, the animals were aerosollychallenged with M catarrhalis strain 25238. Immunization with dLOS—CRMgenerated a significant increase in secreting IgA and IgG in nasalwashes, lung lavage and saliva, and serum IgG, IgM and IgA against LOSof M. catarrhalis as detected by an indirect enzyme-linked immunosorbentassay (ELISA). The dLOS—CRM also elicited LOS-specific IgA, IgG, and IgMantibody-forming cells (AFCs) in different lymphoid tissues as measuredby an enzyme-linked immunospot (ELISPOT) assay. LOS-specific IgA AFCswere found in the nasal passages, spleens, nasal-associated lymphoidtissues (NALT), cervical lymph nodes (CLN), lungs, and small intestines.LOS-specific IgG and IgM AFCs were only detected in the spleens, CLN andnasal passages. Furthermore, the dLOS—CRM vaccine generated asignificant bacterial clearance in the nasopharynx and lungs whencompared to the controls (P<0.01) following an aerosol challenge withthe homologous strain 25238. A comparison of dLOS—CRM, dLOS-TT anddLOS-UspA through intranasal immunization resulted in similar protectionagainst M catarrhalis. Intriguingly, intranasal immunization withdLOS—CRM containing CT showed a higher level of bacterial clearance inboth sites when compared to subcutaneous injections with dLOS—CRM plusCT adjuvant. These data indicate that dLOS—CRM induces specific mucosaland systemic immunity against M catarrhalis through intranasalimmunization, and provides effective bacterial clearance in the mousenasopharynx and lungs. Therefore, it is envisioned as being an efficientroute for vaccines to prevent otitis media and lower respiratory tractinfections caused by M catarrhalis.

[0082] Animals. Female BALB/c mice (6-8 weeks old) were purchased fromTaconic farms Inc. (Germantown, N.Y.).

[0083] Conjugate vaccine. Purification of LOS from M catarrhalis strain25238, detoxification of the LOS, and conjugation of dLOS to carrierprotein including CRM, TT, UspA were performed as described previously(Gu, X. X. et al. 1998 Infect Immun 66:1891-1897).

[0084] LOS purification. Type A strain ATCC 25238 was grown on chocolateagar at 37° C. in 5% CO₂ for 8 h and transferred to 250 ml of 3% trypticsoy broth (Difco Laboratories, Detroit, Mich.) in a 500-ml bottle. Thebottle was incubated at 110 rpm in an incubator shaker (model G-25; NewBrunswick Scientific Co., Edison, N.J.) at 37° C. overnight. The culturewas transferred to six 2.8-liter baffled Fembach flasks, each of whichcontained 1.4 liters of tryptic soy broth. The flasks were shaken at 110rpm and maintained at 37° C. for 24 h. The culture was centrifuged at15,000×g and 4° C. for 10 min to collect the cells. The cell pelletswere washed once with 95% ethanol, twice with acetone, and twice withpetroleum ether (Masoud, H. et al. 1994 Can J Chem 72:1466-1477) anddried to a powder. The LOS was extracted from cells (Gu, X. X. et al.1995 Infect Immun 63:4115 -4120), and the protein and nucleic acidcontents of the LOS were less than 1% (Smith, P. K. et al. 1985 AnalBiochem 150:76-85; Warburg, O., and W. Christian. 1942 Biochem Z310:384-421).

[0085] Detoxification of LOS. Anhydrous hydrazine treatment of LOSremoves esterified fatty acids from lipid A (Gu, X. X. et al. 1996Infect Immun 64:4047-4053; Gupta, R. K. et al. 1992 Infect Immun60:3201-3208). LOS (160 mg) was suspended in 16 ml of anhydroushydrazine (Sigma Chemical Co., St. Louis, Mo.) and incubated at 37° C.for 3 h with mixing. This suspension was cooled on ice and addeddropwise with cold acetone until a precipitate formed. The mixture wascentrifuged at 5,000×g and 5° C. for 30 min. The pellet was washed twicewith cold acetone, dissolved in pyrogen-free water at a finalconcentration of 10 to 20 mg/ml, and then ultracentrifuged at 150,000×gand 5° C. for 3 h. The supernatant was passed through a column (1.6 by90 cm) of Sephadex G-50 (Pharmacia LKB Biotechnology, Uppsala, Sweden)eluted with 25 mM ammonium acetate and monitored with a differentialrefractometer (R-400; Waters, Milford, Mass.). The eluate was assayedfor carbohydrate by a phenol-sulfuric acid method (Dubois, M. et al.1956 Anal Biochem 28:250-256). The carbohydrate-containing fractionswere pooled, freeze-dried, and designated dLOS.

[0086] Derivatization of dLOS. Adipic acid dihydrazide (ADH; AldrichChemical Co., Milwaukee, Wis.) was bound to dLOS to form adipichydrazide (AH)-dLOS derivatives, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS)(Pierce) (Gu, X. X., and C. M. Tsai 1993 Infect Immun 61:1873-1880).dLOS (70 mg) was dissolved in 7 ml of 345 mM ADH (molar ratio of ADH toLOS is ˜100 to 1, based on an estimated M_(r) of 3,000 for dLOS)(Edebrink, P. 1994 Carbohydr Res 257:269-284). Sulfo-NHS was added to aconcentration of 8 mM, the pH was adjusted to 4.8, and EDC was added toa concentration of 0.1 M. The reaction mixture was stirred andmaintained at pH 4.8 for 3 h. The reaction mixture was adjusted to pH7.0 and passed through the G-50 column as described above. The eluatewas assayed for carbohydrate and for AH (Kemp, A. H., and M. R. A.Morgan 1986 J Immunol Methods 94:65-72). The peaks containing bothcarbohydrate and AH were pooled, freeze-dried, and designated AH-dLOS.AH-dLOS was measured for its composition, using dLOS and ADH asstandards (Dubois, M. et al. 1956 Anal Biochem 28:250-256; Kemp, A. H.,and M. R. A. Morgan 1986 J Immunol Methods 94:65-72).

[0087] Conjugation of AH-dLOS to proteins. TT was obtained fromConnaught Laboratories Inc., Swiftwater, Pa., and HMP was purified fromNTHi strain 12 (Barenkamp, S. J. 1996 Infect Immun 64:1246-¹²⁵I).AH-dLOS was coupled to TT or HMP to form conjugates (Gu, X. X., and C.M. Tsai 1993 Infect Immun 61:1873-1880). Briefly, AH-dLOS (30 mg) wasdissolved with 3 ml of water and mixed with 15 mg of TT (5.9 mg/ml) orwith 12 mg of HMP (4 mg/ml). The molar ratio of AH-dLOS to both TT(M_(r), 150,000) and HMP (M_(r), 120,000) was ˜100 to 1. The pH wasadjusted to 5.4, and EDC was added to a concentration of 0.05 to 0.1 M.The reaction mixture was stirred, and the pH was maintained at 5.4 for 3h. The reaction mixture was adjusted to pH 7.0, centrifuged, and passedthrough a column (1.6 by 90 cm) of Sephacryl S-300 in 0.9% NaCl. Peaksthat contained both protein and carbohydrate were pooled and designateddLOS-TT or dLOS-HMP. Both conjugates were analyzed for their compositionof carbohydrate and protein, using dLOS and bovine serum albumin (BSA)as standards (Dubois, M. et al. 1956 Anal Biochem 28:250-256; Smith, P.K. et al. 1985 Anal Biochem 150:76-85).

[0088] Immunization and sample collection. Mice, 6-8 for each group,were immunized intranasally (i.n 4 times, or subcutaneously (s.c 3times, with PBS or 5 μg of dLOS-protein at 1˜2-week intervals,respectively. The total volume of administration is 10 μl for i.n.inoculation, or 0.2 ml for s.c. injection with or without Ribi 700 (25μg/mouse) or cholera toxin (CT, 1 μg/mouse) adjuvant. One week after thelast immunization, nasal washes, saliva, lung lavage, fecal extracts,and sera were collected.

[0089] Detection of LOS-specific antibodies by ELISA. The titers of LOSspecific antibodies in nasal washes, saliva, lung lavage, fecal extractsand sera were determined by ELISA using M catarrhalis strain 25238 LOSas a coating antigen. The antibody endpoint titer was defined as thehighest dilution of sample giving an A405 twofold greater than that ofnegative controls.

[0090] Detection of LOS-specific antibody-forming cells (AFCs).Mononuclear cells were taken from the nasal passage, spleen,nasal-associated lymphoid tissue, cervical lymph node, Peyer's patch andlung. Numbers of LOS-specific IgA-, IgG-, and IgM-producing cells ineach tissue were determined by an enzyme-linked immunospot (ELISPOT)assay.

[0091] Bacterial aerosol challenge. The bacterial aerosol challengeswere carried out one week after the last immunization in an inhalationexposure system (Glas-col, Terre Haute, Ind.) (Hu, W. G. et al. 1999Vaccine 18:799-804). Conditions were as follows: challenge dose ofbacteria, 108 to 5×10⁸ CFU/ml in the nebulizer; nebulizing time, 40 min;vacuum flowmeter, 60 standard ft³/h; and compressed air flowmeter, 10ft³/h.

[0092] Measurement of bacterial clearance from mouse nasopharynx andlungs. At 6 h postchallenge, mice lungs were removed, and homogenated in5 ml of PBS for 1 min at low speed in a tissue homogenizer (StomacherLab System Model 80, Seward, London, UK). At the same time, nasal washeswere obtained by flushing the nasal cavity with 200 μl of PBS. Theappropriately diluted or undiluted lung homogenates, and nasal washeswere plated on chocolate agar plates, and the bacterial colonies werecounted after overnight incubation. In addition, sera, nasal washes andlung homogenates were collected for antibody quantification.

[0093] Statistical analysis. The viable bacteria were expressed as thegeometric mean CFU of n independent observations±standard deviation.Geometric means of reciprocal antibody titers were determined.Significance was determined by Student's t test.

RESULTS

[0094] TABLE 4 Murine antibody responses against LOS of M. catarrhalisstrain 25238 elicited by dLOS-CRM conjugate vaccine ImmunizationAntibody GM antibody ELISA titers^(b) Group^(a) class Nasal wash Lunglavage Saliva Fecal Extract Serum {circle over (1)}dLOS-CRM IgA 169(36-782)**^(c) 144 (41-501)** 30 (7-124)** 21 (3-159)* 48 (14-167)** IgG14 (6-31)** 26 (8-83)** 3 3 56 (8-412)** IgM 3 3 3 3 48 (14-167)**{circle over (2)} dLOS IgA 26 (13-53)** 5 (2-13) 5 (3-9) 3 10 IgG 3 3 33 10 IgM 3 3 3 3 26 (18-39)** {circle over (3)} PBS IgA 3 3 3 3 10 IgG 33 3 3 10 IgM 3 3 3 3 10

[0095] TABLE 5 Effect of intranasal immunization with dLOS-CRM onbacterial recovery of homologous strain 25238 in mouse nasopharynx andlungs^(a) Nasopharynx Lung Bacterial Bacterial Bacterial Bacterialrecovery^(b) reduction^(c) recovery^(b) reduction^(c) Immunogen(CPU/lung) (%) (CFU/mouse) (%) {circle over (1)}dLOS-CRM  91 75 1290 87(41-201)^(d) (555-2998)^(d) {circle over (2)} dLOS 354 2 8872 8(188-669) (6468-12169) {circle over (3)} PBS 362 0 9656 0 (250-516)(8130-11472)

[0096] TABLE 6 Murine antibody responses against LOS of M. cat strain25238 elicited by different dLOS-protein conjugates ImmunizationAntibody GM antibody ELISA titers^(b) Group^(a) class Nasal wash Lunglavage Saliva Fecal Extract Serum {circle over (1)}dLOS-CRM IgA 231(61-877)**^(c) 105 (24-462)** 12 (4-32)** 5 (2-12) 56 (24-133)** IgG 16(9-29)** 26 (7-97)** 4 (2-6) 5 (3-10) 123 (36-418)** IgM 3 13 (6-32)** 33 30 (12-74)** {circle over (2)} dLOS IgA 103 (35-307)** 52 (10-274)** 5(2-13) 5 (2-11) 34 (14-86)** IgG 11 (4-31)** 10 (3-32)* 4 (2-9) 4 (2-9)45 (20-103)** IgM 3 7 (4-13)** 3 3 20 (9-45)* {circle over (3)}dLOS-UspA IgA 26 (10-65)** 17 (10-31)** 4 (2-7) 5 (2-11) 17 (10-31)* IgG6 (3-15)** 6 (3-12)** 3 6 (2-24) 20 (11-35)** IgM 3 5 (3-10)* 3 3 17(10-31)* {circle over (4)} PBS IgA 3 3 3 3 10 IgG 3 3 3 3 10 IgM 3 3 3 310

[0097] TABLE 7 Murine antibody responses against LOS of M. catarrhalisstrain 25238 elicited by dLOS-CRM conjugate through differentimmunization regimens Immunization Antibody GM antibody ELISA titers^(b)Immunogen^(a) route class Nasal wash Lung homogenate Serum {circle over(1)}dLOS-CRM intranasal IgA 118 (26-545) 111 (23-530) 78 (18-348) IgG 20(5-73) 73 (13-396) 156 (20-1192) IgM 3 31 (11-87) 26 (13-53) {circleover (2)} PBS intranasal IgA 3**^(c) 3** 10** IgG 3** 3** 10** IgM 3 3**10** {circle over (3)} dLOS-CRM subcutaneous IgA 13 (8-22)** 15 (7-31)**17 (8-40)* IgG 15 (9-27) 161 (22-1185) 536 (60-4805) IgM 3 31 (11-87) 17(8-40) {circle over (4)} PBS subcutaneous IgA 3** 3** 10** IgG 3** 3**10** IgM 3 3** 10**

[0098] Conclusions. Intranasal immunization with dLOS—CRM induced bothmucosal and systemic immunity (Table 4, FIGS. 8A-C). Intranasalimmunization with dLOS—CRM significantly enhanced M catarrhalisclearance from mouse nasopharynx and lungs (Table 5). Differentconjugate vaccines elicited similar protection against M catarrhalis(Table 6, FIGS. 9A-C and 10A-B). Compared to subcutaneous injection,intranasal immunization with dLOS—CRM showed a higher level of bacterialclearance from mouse nasopharynx and lungs (Table 7, FIGS. 11 and 12).At each time point, immunized mice significantly reduced bacterialrecovery from nasopharynx and lungs, and bacterial recovery becameundetectable within 24 h postchallenge (FIGS. 13 and 14).

[0099] While the present invention has been described in some detail forpurposes of clarity and understanding, one skilled in the art willappreciate that various changes in form and detail can be made withoutdeparting from the true scope of the invention. All references referredto above are hereby incorporated by reference.

What is claimed is:
 1. An aerosolizer for intranasal administration ofan immunogenic composition comprising an immunizing amount ofNontypeable Haemophilus influenzae (NTHi) or Moraxella catarrhalislipooligosaccharide (LOS) from which at least one primary O-linked fattyacid has been removed to form detoxified LOS (dLOS) and an immunogeniccarrier covalently linked thereto, optionally wherein said dLOS and saidimmunogenic carrier are covalently linked by a linker, and a mucosaladjuvant or delivery system.
 2. A method for inducing an immunologicalresponse comprising intranasal administration of an immunogeniccomposition comprising an immunizing amount of Nontypeable Haemophilusinfluenzae (NTHi) or Moraxella catarrhalis lipooligosaccharide (LOS)from which at least one primary O-linked fatty acid has been removed toform detoxified LOS (dLOS) and an immunogenic carrier covalently linkedthereto, optionally wherein said dLOS and said immunogenic carrier arecovalently linked by a linker, and a mucosal adjuvant or deliverysystem, whereby colonization by NTHi or M catarrhalis is inhibited orotitis media or other respiratory disease caused by NTHi or Mcatarrhalis infection is prevented.
 3. The aerosolizer or method ofclaims 1 or 2, wherein said mucosal adjuvant or delivery systemcomprises aluminum salts.
 4. The aerosolizer or method of claims 1 or 2,wherein said mucosal adjuvant or delivery system comprises chitosan. 5.The aerosolizer or method of claims 1 or 2, wherein said mucosaladjuvant or delivery system comprises cytokines.
 6. The aerosolizer ormethod of claims 1 or 2, wherein said mucosal adjuvant or deliverysystem comprises saponins.
 7. The aerosolizer or method of claims 1 or2, wherein said mucosal adjuvant or delivery system comprises muramyldipeptide (MDP) derivatives.
 8. The aerosolizer or method of claims 1 or2, wherein said mucosal adjuvant or delivery system comprises CpGoligos.
 9. The aerosolizer or method of claims 1 or 2, wherein saidmucosal adjuvant or delivery system comprises lipopolysaccharide (LPS)of gram-negative bacteria.
 10. The aerosolizer or method of claims 1 or2, wherein said mucosal adjuvant or delivery system comprisesmonophosphoryl lipid A (MPL)
 11. The aerosolizer or method of claims 1or 2, wherein said mucosal adjuvant or delivery system comprisespolyphosphazenes.
 12. The aerosolizer or method of claims 1 or 2,wherein said mucosal adjuvant or delivery system comprises emulsions.13. The aerosolizer or method of claims 1 or 2, wherein said mucosaladjuvant or delivery system comprises virosomes.
 14. The aerosolizer ormethod of claims 1 or 2, wherein said mucosal adjuvant or deliverysystem comprises Iscoms.
 15. The aerosolizer or method of claims 1 or 2,wherein said mucosal adjuvant or delivery system comprises cochleates.16. The aerosolizer or method of claims 1 or 2, wherein said mucosaladjuvant or delivery system comprises poly(lactide-co-glycolides) (PLG)microparticles.
 17. The aerosolizer or method of claims 1 or 2, whereinsaid mucosal adjuvant or delivery system comprises poloxamer particles.18. The aerosolizer or method of claims 1 or 2, wherein said mucosaladjuvant or delivery system comprises virus-like particles.
 19. Theaerosolizer or method of claims 1 or 2, wherein said mucosal adjuvant ordelivery system comprises heat-labile enterotoxin (LT) B subunit. 20.The aerosolizer or method of claims 1 or 2, wherein said mucosaladjuvant or delivery system comprises cholera toxin (CT) B subunit. 21.The aerosolizer or method of claims 1 or 2, wherein said mucosaladjuvant or delivery system comprises mutant toxins.
 22. The aerosolizeror method of claims 1 or 2, wherein said mucosal adjuvant or deliverysystem comprises microparticles.
 23. The aerosolizer or method of claims1 or 2, wherein said mucosal adjuvant or delivery system comprisesliposomes.